The long term goals of the Principal Investigator are to investigate the changes in the tau molecule that lead to a decrease in its normal functions and increase its pathological ability to self-assemble into filaments found in many neurodegenerative disorders. The purpose of this proposal is to determine the effects of changes in phosphorylation that are observed in Alzheimer's disease (AD) on the functions of the tau protein. These experiments will have an immediate impact in AD research since there is a current controversy over the role of phosphorylation and "hyperphosphorylation" in the polymerization of tau into filaments found in AD pathology. We hypothesize that site-specific phosphorylation events can change the conformation of tau and create variants of the molecule that have an increased ability to polymerize, a decreased ability to bind to and stabilize microtubules, or both. We also hypothesize that the phosphorylation of the tau molecule will increase the stability of tau filaments, which could account for their longevity in affected neurons in AD. We will test these hypotheses by accomplishing the following specific aims: 1) The effects of site-specific phosphorylation events found in AD on the polymerization of the longest isoform of tau and the stability of those filaments will be assessed using an in vitro polymerization paradigm monitored by laser light scattering and quantitative electron microscopy. 2) The effects of AD-like phosphorylation on the ability of tau to bind to and stabilize microtubules will be assessed using tubulin binding assays and microtubule polymerization assays in the presence of phospho-variants of tau. 3) The effects of site-specific phosphorylation events will be modeled in the background of the six major isoforms of tau since these isoforms can be differentially involved in neurodegenerative diseases. 4) The effects of "hyperphosphorylation" will be modeled to determine the effects of multiple phosphorylation events on the polymerization and stability of tau filaments. 5) The effects of phosphorylation that occur after tau polymerization on the stability of tau filaments will be investigated using quantitative electron microscopy.